Title: Beta cell ontogenesis and the insulin production apparatus
Authors: R.Scott Heller and Ole D. Madsen, Hagedorn Research Institute, Gentofte, Denmark
Abstract
The pancreatic insulin producing beta cell is a highly specialized cell that develops into the
main endocrine cell types in the Islets of Langerhans of the pancreas from the primitive gut endoderm.
A large number of specific transcription factors have been demonstrated to be crucial to the
development and function of this highly specialized cell. Recent, genome wide association scans as
well as study of maturity onset diabetes of the young genes has demonstrated that most of these
genes are expressed in the pancreatic beta cell and are involved in not only transcriptional functions
but also the insulin secretory apparatus. This chapter provides a short overview of these subjects.
Early pancreatic organogenesis
Pancreas organogenesis is a largely conserved process throughout vertebrate development.
Phylogenetic studies of pancreas (Heller 2010) suggest that the insulin-producing beta cell founded
the pancreatic organ (together with few somatostatin-producing cells and cytokeratin immunoreactive
cells (Christensen, Madsen and Heller- unpublished data) and the alpha cells, acinar cells and the PP
cells entered at later stages (Falkmer 1995, Madsen 2007) (Figure 1). Shortly after gastrulation and
formation of the endoderm both ventral and dorsal regions initiate pancreas formation (pancreatic
anlage) ((Zaret & Grompe 2008) – for review).The ventral pancreatic bud becomes the head of the
pancreas while the dorsal bud becomes the tail. In certain fish species, such as salmonoid fish, the
dorsal part primarily forms a giant islet structure, - the Brockmann Body (which become embedded in
ventrally derived pancreatic exocrine parenchyma). Ventral and dorsal origin of pancreas is likely
governed by distinct mechanisms of specification (Spence et al 2009) and different cues may appear
to control the subsequent development of pancreatic tissue that otherwise appears almost identical in
the head and tail regions – maybe with the exceptions of the islet composition where more pp-rich islet
appear in the head region in contrast to more alpha-cell rich islet in the tail region. Pdx-1 is first
detected in the earliest pancreatic anlage (i.e. at e8.5 – e9 in dorsal and ventral buds of the mouse
pancreas)(Jorgensen et al 2007). Its expression subsequently expands to comprise duodenal and
antral stomach tissues. Pdx-1 deficiency causes pancreas agenesis (in zebrafish, mouse, and man)
(Jonsson et al 1994, Offield et al 1996, Stoffers et al 1997). Subsequent to the onset of Pdx-1
expression two additional transcription factors become activated including Nkx6.1 (restricted the beta
cells in the adult pancreas) and Ptf1a (restricted to the acinar cells of the adult pancreas). At this stage
most of the cells in the buds co-express all 3 factors (Hald et al 2008) – and such cells are considered
to represent multipotent pancreatic progenitors (See Figure 1) that subsequently can give rise to all
mature pancreatic cell types (Burlison et al 2008, Zhou et al 2007). A new role of sox17 was recently
reported by the Wells research group. Importantly, they are able to demonstrate that Sox17 is critical
for the proper segregation of Pdx-1 progenitors to the ventral pancreas and not the liver or biliary tract
(Spence et al 2009).
Expansion of progenitors
While the early buds still form in the absence of Pdx-1 (Jonsson et al 1994, Offield et al 1996) they fail
to proliferate and the Pdx-1-null phenotype is reminiscent of that of FGF10-nulls (Bhushan et al 2001)
suggesting that FGF10 is responsible for the proliferation of the earliest pancreatic progenitors.
Profound expansion of the triple-positive cells leads to the formation of a multilayered “squamous”
epithelium where luminal cavities and polarization of epithelial cells commence (Kesavan et al 2009).
Concomitantly the process of branching morphogenesis characterizes the following period of pancreas
development where true epithelial layer form and expands by branching. During branching
morphogenesis there is a stringent segregation of the expression domains for Nkx6.1 and Ptf1a such
that Nkx6.1 remains within the trunks of the branches while Ptf1a dominates in the tips (Hald et al
2008). The endocrine cells will subsequently arise in the trunk – domain (dependent on Ngn3
activation (Gradwohl et al 2000, Gu et al 2002) while the tip of the branches will form the acini (Zhou
et al 2007)
Early differentiation
Pancreatic endocrine differentiation in rodents is described to occur in two phases – the
primary and the secondary transition (Jorgensen et al 2007, Pictet & Rutter 1972). During the primary
transition mature (based on EM morphology) alpha cells are formed in readily detectable numbers.
These early glucagon cells express the prohormone converting enzymes PC1/3 in addition to PC2
(Lee et al 1999, Wilson et al 2002) – in contrast to the adult alpha cell (only expressing PC2, required
for glucagon processing (Furuta et al. 2001). As a consequence the early glucagon cells produce
glucagon as well as Glp1 and Glp2 (Kreymann et al 1991). It is plausible that early glucagon cells later
down-regulate expression of PC1/3 and contribute to the adult type alpha cells in the mature islets.
Also early insulin gene activity is measurable as mRNA and immunoreactive insulin during the primary
transition. Early reports on the existence of early multi-hormonal endocrine cells co-expressing
glucagon and low levels of insulin suggested the existence of a multipotent endocrine multi-hormonal
progenitor. Elegant lineage tracing studies by Herrera demonstrated that insulin and glucagon cells
arise from distinct lineages that diverge prior to the onset of hormone gene expression (Herrera et al
2002). The nature of double hormone positive cells during development remains elusive. Beta cells
primarily arise during the secondary transition (e13-15) as described below and occur in the trunk
region characterized by Nkx6.1 and Pdx-1 expression (Jorgensen et al 2007).
The Choice to become a -cell
Once the endocrine precursor cells (Ngn3+) progress beyond a very early phase, a serious
choice is forced upon them. What endocrine cell type will I become: Insulin, glucagon, somatostatin,
pancreatic polypeptide, or ghrelin? Recent evidence suggests that this is already pre-determined
(Desgraz & Herrera 2009), while others support a model where a balance between Pax4 and Arx is
the most critical decision on what will turn the Ngn3+ cell into either  or  cells (Kordowich et al 2010).
Very eloquent in vivo clonal analysis experiments of Ngn-3 expressing cells, demonstrated
that every Ngn3 cell becomes one endocrine cell type with a restricted and specific differentiation
potential that is determined at a very early stage (Desgraz & Herrera 2009). A future perspective is to
better understand if there is a certain gene expression profile that marks cells for a specific fate prior
to Ngn3 activation or if it is the ability of that single cell to respond to specific extracellular cues that
drives this progression to becoming a -cell. Understanding these things will be crucial to directed
differentiation of stem cells to pancreatic -cells.
The importance of the Arx and Pax4 transcription factors in cell fate decisions have been well
elucidated in the past 13 years since the publication of the Pax4 knockout mouse (Sosa-Pineda et al
1997). Arx promotes the glucagon/pancreatic polypeptide cell fates, while Pax4 induces insulin and
somatostatin cell fates (Collombat et al 2005, Collombat et al 2007, Collombat et al 2003, Collombat et
al 2009, Kordowich et al 2010). In a series of very well conducted experiments Collombat and
colleagues have been able to demonstrate that Pax4 is not absolutely required to specify the
somatostatin and insulin cell fates but acts by inhibiting the glucagon cell fate, which studies have
shown to be the first hormone cell type (or default) that is created in the genesis of endocrine cells
(Jorgensen et al 2007). Additionally, Pax4 is able to direct endocrine cells into the beta cells, even
mature glucagon cells (Collombat et al 2009, Liu & Habener 2009).
Young - cells
Once high level Nkx6.1 and Pdx-1 expression is observed, this is a strong sign that an
endocrine cell is committed to become a -cell. A number of specific transcription factors (IA-1,
Nkx2.2, Pax6) are important for the -cell identity and mutations in these create endocrine cells
without the expression of insulin. IA-1 is a direct target of Ngn3 and has been shown to be necessary
but not sufficient for endocrine cells. Mice lacking IA-1 still have endocrine cells but most are lacking
hormones, while overexpression studies alone do not induce endocrine cell formation (Gierl et al
2006, Mellitzer et al 2006).
Nkx6.1, expressed broadly in the developmental pancreatic trunk epithelium and it specifically
plays a role in the in the mature -cells, as knockout of the gene has a phenotype where mice are
devoid of late but not early created -cells (Sander et al 2000). Pax4 is a very important factor in
promoting the -cell fate. While Pax4 is not directly required to specify the -cell but rather blocks the
-cell fate, it has powerful effects in endocrine precursors to drive -cell differentiation (Collombat et al
2009, Kordowich et al 2010). Nkx2.2 also specifically effects the -cell fate and Pax4-Nkx2.2 double
knockout mice have the same -cell phenotype, which suggests that Nkx2.2 is acting upstream of
Pax4 (Prado et al 2004, Sussel et al 1998).
The earliest -cells found in late embryonic life and early post-natal development is
characterized by the ability to proliferate, a feature that becomes severely decreased just after
weaning in animals. Thereafter, the beta cell mass is maintained by a very slow level of replication
(Granger & Kushner 2009). In the recent in vivo clonal analysis paper, following Ngn3 cell fates, it was
also recognized that -cells proliferate at a very slow rate and that the number of islets remains
constant during 2-10 months of life (Desgraz & Herrera 2009). This has also been demonstrated in
Ob/Ob mice (Bock et al 2003).
Mature -cells
Once the -cell has matured it is an extremely metabolically active cell which has to precisely
function to deliver insulin in direct response to the blood glucose levels to maintain glucose
homeostasis within a very precise range. The insulin apparatus has been honed after many millions of
years of evolution. Many transcription factors have been shown to be very important in the regulation
of glucose stimulated insulin secretion and these include MafA, FoxO1, Pdx-1 and others (see recent
review by (Shao et al 2009). Furthermore, the demonstration that so many single mutations in -cell
genes can directly disrupt the function only highlights the importance of the high demands and precise
regulation that is required in the -cell.
The ability of the -cell to adapt to physiological and patho-physiological conditions such as
pregnancy and obesity, increases the demand for insulin. The -cell under normal circumstances
responds with increased biosynthesis of insulin and increased replication of -cell numbers. Peripheral
insulin resistance (obesity) triggers a compensatory upregulation of beta cell mass (Matveyenko &
Butler 2008, Rahier et al 2008). In mice this process appears to be driven via the insulin receptor on
the beta cell (Assmann et al 2009) and requiring IRS2 to mediate the mitotic signal (Withers et al
1998). Insulin itself is thus an obvious candidate as a positive feed-back growth-signal for the beta
cells, which make sense as long as glucose above near-normal range.
Maturity onset diabetes of the young (MODY) is described as having the following
characteristics: a primary defect in insulin secretion and hyperglycemia, monogenic autosomal
dominant mode of inheritance, age at onset less than 25 years, and the lack of auto-antibodies.
Mutations in six genes have been described and these include the enzyme glucokinase, which causes
MODY2, and the transcription factors HNF-4 alpha, TCF1, Pdx-1, TCF2, and NeuroD1, which cause
MODY1, 3, 4, 5, and 6, respectively. Most recently, KLF11, has been described as MODY7 and is a
novel p300-dependent regulator of Pdx-1 (MODY4) transcription in pancreatic islet beta cells
(Fernandez-Zapico et al 2009). One thing that all these genes have in common is their expression in
the mature -cell.
Pdx-1 is a critical transcription factor in the adult -cell and haplodeficieny in mice and humans
leads to diabetic phenotypes. A recent study by Stoffers research group has highlighted an important
new role for Pdx-1 in the regulation of -cell endoplasmic reticulum stress responses, where it directly
regulates a number of these genes and processes (Sachdeva et al 2009).
Recent genome wide association (GWA) scans of the human genome have identified over 20
new genes or genomic sites associated with type II diabetes (Staiger et al 2009). A number of these
genes are also expressed and act in the insulin producing -cell, with TCF7L2, being the best
characterized (Grant et al 2006, Liu & Habener 2010). In addition, mutations in SLC30A3 and ZnT8,
both which effect zinc transport, an important part of the insulin secretory granule, have been identified
and shown to have important roles in beta cells (Nicolson et al 2009, Smidt et al 2009).
These data further highlight the complexity of the pancreatic beta cell and how precise
regulation of the beta cell is so important especially under physiological stress such as diabetes.
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